Research addressing the main specific aims of this project focused on cellular and circuit mechanisms generating the respiratory rhythm and neural activity patterns in the brainstem of rodents. Experimental studies were performed with isolated in situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal or mature rats and mice. Previously we have identified the brainstem locus, called the pre-Botzinger complex (pre-BotC), that contains populations of neurons critical for respiratory rhythm generation. We have further exploited methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing structural imaging performed simultaneously with functional activity imaging by multi-photon laser scanning microscopy of the neurons labeled with fluorescent genetically-encoded calcium sensor and/or fluorescent proteins. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of expression of neuron membrane channels, receptors, and neurotransmitter-related proteins. With these approaches, we have performed high-resolution spatio-temporal imaging of neuron activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-BotC in vitro. These studies have provided the most direct experimental evidence to date that rhythm generation involves an excitatory network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. By applying optogenetic approaches we have established that a critical population of glutamatergic neurons with voltage-dependent oscillatory properties is the substrate for inspiratory rhythm generation in the pre-BotC. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-BotC, including with intracellular recording techniques in situ and advanced electrophysiolgical approaches such as the dynamic clamp applied in vitro, continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratory rhythm in the intact mammalian nervous system. Studies in progress based on intracellular recording approaches applied in situ are analyzing in detail how distinct populations of excitatory and inhibitory neurons interact to generate the respiratory rhythm and pattern as well as to test predictions of our network models. Furthermore, our new optogenetics-based studies involving photo-inhibition or photo-excitation of inhibitory respiratory neurons have established a fundamental role of inhibitory microcircuits including in the pre-BotC in respiratory pattern generation. Other studies have provided additional evidence that neuronal persistent sodium currents and several types of leak or background conductances represent critical ionic conductance mechanisms for generation and control of respiratory oscillations. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons in vitro, as well as our current immunohistochemical and pharmacological studies, have identified a specialized set of transient receptor potential (TRP) cationic channels that also represent important regulators of neuron excitability and current studies are directed toward understanding how these channels may contribute to electrophysiological behavior of respiratory circuit neurons. Other electrophysiological studies have demonstrated that leak conductance mechanisms are critically involved in the regulation of rhythmic breathing patterns by a diverse set of endogenous neurochemicals that modulate these conductances as well as by physiological control signals including carbon dioxide and oxygen. In addition, we have conducted novel studies of the role of astrocytes in modulatory control of neural circuit activity in the pre-BotC, including by the release of signaling molecules such as ATP, which is hypothesized to excite the rhythm generating neurons, in response to elevated carbon dioxide (hypercapnia) or reduced oxygen (hypoxia) in vivo. We have determined by employing viral-vectors that selectively interfere with release of glial transmitters or disrupt ATP-mediated signaling that astrocytes respond to hypercapnia and hypoxia in vivo to regulate the activity of pre-BotC circuits to homeostatically adjust the breathing frequency to partially compensate for these physiological disturbances. In our previous studies employing novel pharmacogenetic approaches applied in situ and in vivo, neurons of the retrotrapezoid nucleus (RTN) that have chemosensory properties were also shown to provide a critical excitatory modulatory input to core components of the respiratory network including the pre-BotC to regulate generation of inspiratory neural activity. Our new studies showing involvement of astrocytes in chemosensory regulation at the level of the pre-BotC have led us to propose new conceptual models for the physiological regulation of key respiratory circuits that incorporate multiple neuromodulatory control mechanisms including astrocytic mechanisms. We are currently extending our optogenetics-based studies to manipulate activity of regionally specific neuronal and astrocyte populations to further investigate how these different populations contribute to generation and control of respiratory neural activity in various (patho)physiological states.